Edexcel International A Level Biology Past Paper PDF

Head to www.savemyexams.com for more awesome resources Edexcel International A Level Your notes Biology Microbiology Contents 6.1 Culturing Microorganisms 6.2 Measuring the Growth of Microorganisms 6.3 The Bacterial Gro...

Head to www.savemyexams.com for more awesome resources Edexcel International A Level Your notes Biology Microbiology Contents 6.1 Culturing Microorganisms 6.2 Measuring the Growth of Microorganisms 6.3 The Bacterial Growth Curve 6.4 Core Practical 13: Rate of Growth of Microorganisms 6.5 Comparison of Bacterial & Viral Structure Page 1 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources 6.1 Culturing Microorganisms Your notes Culturing Microorganisms Most microorganisms are only visible using a microscope For microbe investigations it is therefore necessary to culture microorganisms This grows enough microorganisms to make measurements during investigations E.g. bacteria reproduce by cloning themselves, so when they are grown on agar gel they form a colony of identical individuals that is visible to the naked eye Microorganisms must be provided with everything they need to grow such as Nutrients Oxygen Note that anaerobic microorganisms would require the absence of oxygen Optimum pH Favourable temperature Microorganisms should be cultured with great care There is always the risk that a mutation could lead to the formation of pathogenic strains Pathogenic bacteria from the environment could contaminate the bacterial culture being investigated Remember to Follow health and safety precautions Ensure that all equipment are sterilised before culturing the bacteria Sterilising involves killing microorganisms, e.g. by heating to a high temperature or the use of antimicrobial chemicals Keep the culture in the laboratory Seal cultures in a plastic bag and sterilise at high temperature and pressure before disposal Culturing steps Obtain a supply of the type of microorganism to be cultured Provide them with the correct type of nutrients to facilitate growth A nutrient growth medium (plural media) containing carbon, nitrogen, and minerals is typically used The medium could be in the form of a liquid culture or a solid nutrient agar, a type of gel extracted from seaweed Ensure that the nutrient medium is kept under sterile conditions until use By adjusting the type of nutrients in the medium, conditions will be created for the optimal growth of a certain type of microorganism; this is known as a selective medium Selective media can be used to identify mutant strains of microorganisms or those that are resistant to antibiotics They are also useful for identifying genetically modified microorganisms Microorganisms are introduced to a growth medium using inoculation with a sterilised inoculation loop Inoculation can be used to transfer microorganisms between media, e.g. Page 2 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources From agar gel into a liquid culture flask From a liquid culture flask onto agar gel The new medium should be sealed or covered to avoid contamination from microorganisms in the air; Your notes if growing aerobic microorganisms any seal or cover should not be airtight Flasks can be sealed with a sterile cotton wool stopper Petri dishes can be covered with a lid Label the medium clearly and incubate at around 20 °C to prevent the growth of pathogenic microorganisms Microorganisms that are pathogenic to humans will grow best at around 37 °C In a hospital or research laboratory a higher temperature might be used to obtain faster results Page 3 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources Your notes Page 4 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources Your notes A metal inoculating loop can be used to transfer microorganisms from a liquid broth medium onto an agar gel medium. A Bunsen burned enables sterilisation of the loop between uses. Growing a single type of microorganism In order to grow a single type of microorganism, or a pure culture, the specific microorganism must be isolated This can be done by using knowledge about the needs of the microorganism to be cultured or those of microorganisms that may contaminate the culture Examples include Growing the culture under either aerobic or anaerobic conditions to reduce the variety of microorganisms in the culture Using a selective medium that is tailored to the specific requirements of the desired microorganism Indicator media can provide a colour change to distinguish desired colonies from the rest Being able to isolate pathogenic microorganisms is useful in the diagnosis and treatment of diseases Page 5 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources 6.2 Measuring the Growth of Microorganisms Your notes Measuring the Growth of Microorganisms Bacteria and most other microorganisms are too small to count with the naked eye There are a variety of methods that can be used to count microorganisms and it is important to be able to make an appropriate choice when conducting investigations These methods include Cell counts Dilution plating Measuring area and mass Optical methods Cell counts A microscope and haemocytometer can be used to count single-celled microorganisms A haemocytometer is a microscope slide with a rectangular chamber that is marked with grid lines The chamber can hold a standard volume of 0.1 mm3 Haemocytometers were originally used to count blood cells Haemo = blood Counting cells using a haemocytometer involves the following A nutrient broth is diluted with an equal volume of trypan blue This is a dye that will stain dead cells blue It enables the investigator to only count the living cells The chamber of the haemocytometer is filled with the stained nutrient broth The number of living cells in the four corner squares of the grid are counted Each corner square consists of 16 smaller squares Consistency needs to be used when deciding whether to count cells that are on the lines that border the corner squares E.g. Counting cells on the top and right-hand borders and ignoring cells on the bottom and left-hand borders The mean number of cells from the four corner squares can be calculated The haemocytometer is calibrated to allow the calculation of the number of cells in a known volume of broth Because the haemocytometer chamber can hold exactly 0.1 mm3 of liquid, it is possible to estimate of the cell count in 1 ml of nutrient broth using the following calculation No. of cells per ml nutrient broth = mean cell count x dilution factor x 104 Multiplying by the dilution factor enables calculation of the bacterial cell count in the original broth rather than the diluted broth The multiplication by 104 enables calculation of the bacterial cell count in 1 ml rather than in 0.1 mm3 1 ml = 1 cm3 1 cm3 = 0.1 mm3 x 10 000 10 000 = 1 x 104 Page 6 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources E.g. in a scenario where a nutrient broth is diluted by a factor of 100 and the four corner grid squares contain 20, 14, 19, and 16 cells This gives a mean cell count of 17.25 Your notes No. cells per ml nutrient broth = 17.25 x 100 x 104 = 17 250 000 The living cells in the corner squares of a haemocytometer grid can be counted to determine the number of microorganisms in a standard volume of nutrient broth Dilution plating This method can be used to determine the total viable cell count in a nutrient broth Page 7 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources The nutrient broth is transferred to agar where the bacteria use nutrients in the agar gel to reproduce A single cell that lands on agar reproduces by cloning itself, resulting in a mass of identical cells Your notes known as a colony Each microbial colony that grows on agar gel originated with one viable microorganism, so can be counted as one viable cell Individual microbial colonies can be difficult to identify on an agar plate as they tend to form one large mass This problem can be overcome by diluting the original cultures before transferring the samples to agar; this reduces the number of cells in the original sample so that individual colonies are visible on an agar plate This is why the technique is known as dilution plating To calculate the total viable cell count, the number of colonies are multiplied by the dilution factor A mean can be determined if more than one plate is used Page 8 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources Your notes Dilution plating can provide a way to determine the total viable cell count of a microbial culture Area and mass of fungi Fungi do not always live as single-celled organisms, but can form a mass of elongated cells known as a fungal mycelium (plural mycelia); this means that the methods described above can be unsuitable for measuring the growth of some fungi Measuring the diameter of individual areas of the mycelium can be used to determine the growth of fungi Petri dishes of agar are inoculated with fungal spores and incubated at a suitable temperature The resulting areas of fungal mycelia are then measured This can be used to compare growth rates in different conditions, e.g. at different temperatures The larger the mean diameter, the greater the growth of the fungi Testing the dry mass of fungi is another effective way to measure fungal growth A liquid nutrient broth is inoculated with fungal spores Samples of the nutrient broth are removed at set time intervals The fungal mycelia are removed by filtering or centrifugation The material is dried in an oven overnight and its mass measured The higher the mass, the more fungal growth has occurred Optical methods Turbidimetry is a specialised form of colorimetry that can be used as an alternative method to measure the number of cells in a sample Turbidity is a measure of how cloudy a solution is More turbid = more cloudy Less turbid = less cloudy Colorimetry uses a machine called a colorimeter to shine a beam of light at a sample and measure the amount of light that is either transmitted through or absorbed by the sample The higher the number of cells the more turbid the solution becomes More turbid solutions will absorb more light and allow less light through; this can be measured by a colorimeter This provides an indirect measure of the number of microorganisms present Page 9 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources A calibration curve can be constructed by measuring the turbidity of a series of control cultures while also counting the cells in each culture using a haemocytometer; the results are plotted in a graph of turbidity against cell count Your notes This curve can then be used to estimate the cell count of unknown samples by measuring their turbidity and then reading their cell count from the graph A colorimeter can be used to determine the turbidity of a solution containing microorganisms. The solution containing bacteria is placed into a container called a cuvette and the amount of light that can pass through, or is absorbed by, the solution can be measured. Page 10 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources 6.3 The Bacterial Growth Curve Your notes The Bacterial Growth Curve Bacteria divide using the process of binary fission during which one cell will divide into two identical cells The process is as follows The single, circular DNA molecule undergoes DNA replication Any plasmids present undergo DNA replication The parent cell divides into two cells, with the cytoplasm roughly halved between the two daughter cells The two daughter cells each contain a single copy of the circular DNA molecule and a variable number of plasmids Page 11 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources Your notes Page 12 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources Your notes The process of binary fission produces two identical daughter cells Bacterial growth curve The growth of a bacterial population follows a specific pattern over time; this is known as a growth curve There are 4 phases in the population growth curve of a microorganism population Lag phase The population size increases slowly as the microorganism population adjusts to its new environment and gradually starts to reproduce Exponential phase With high availability of nutrients and plenty of space, the population moves into exponential growth; this means that the population doubles with each division This phase is also known as the log phase Stationary phase The population reaches its maximum as it is limited by its environment, e.g. a lack of resources and toxic waste products. During this phase the number of microorganisms dying equals the number being produced by binary fission and the growth curve levels off Death phase Due to lack of nutrients and a build up of toxic waste build up, death rate exceeds rate of reproduction and the population starts to decline This phase is also known as the decline phase Page 13 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources Your notes There are four phases in the standard growth curve of a microorganism Using logarithms in growth curves During the exponential growth phase bacterial colonies can grow at rapid rates with very large numbers of bacteria produced within hours Dealing with experimental data relating to large numbers of bacteria can be difficult when using traditional linear scales There can be a wide range of numbers reaching from single figures into millions This makes it hard to work out a suitable scale for the axes of graphs Logarithmic scales can be very useful when investigating bacteria or other microorganisms The numbers in a logarithmic scale represents logarithms, or powers, of a base number If using a log10 scale, in which the base number is 10, the numbers on the y-axis represent a power of 10, e.g. 1=101 (10), 2=102 (100), 3=103 (1000) etc. Logarithmic scales allow for a wide range of values to be displayed on a single graph For example, if yeast cells were grown in culture over several hours the number of cells would increase very rapidly from the original number of cells present The results from such an experiment are shown in the graph below using a log scale The number of yeast cells present at each time interval was converted to a logarithm before being plotted on the graph This can be done using a log function on a calculator The log scale is easily identifiable as there are not equal intervals between the numbers on the y- axis Page 14 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources The wide range of cell numbers fit easily onto the same scale Your notes When a log10 scale is used, the scale increases by a factor of 10 each time; this allows large increases in numbers to be shown on a single graph Examiner Tip You won’t be expected to convert values into logarithms or create a log scale graph in the exam. Instead you might be asked to interpret results that use logarithmic scales or explain the benefit of using one! Remember that graphs with a logarithmic scale have uneven intervals between values on one or more axes. Exponential growth rate constants To calculate the number of bacteria in a population the following formula can be used N t = N 0 x 2kt Where Nt = the number of organisms at time t Page 15 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources N0 = the number of organisms at time 0 k = the exponential growth rate constant t = the time for which the colony has been growing Your notes To use this equation the exponential growth rate constant k must be calculated This refers to the number of times the population doubles in a given time period The following formula can be used to calculate the exponential growth rate constant log 10 N t − log 10 N 0 k = log 10 2 x t Worked example A bacterial colony started with 2 individuals and after 3 hours of growing there were 926 bacteria in the colony. 1. Calculate the exponential growth rate constant of this colony 2. Calculate the number of bacteria in the colony after 5 hours Step 1: Calculate the exponential growth rate constant log 10 N t − log 10 N 0 k = log 10 2 x t log 10 ( 926) − log 10 (2) k = log 10 2 x 3 2. 67 k = 0. 30 x 3 k = 3 Step 2: Calculate the number of bacteria after 5 hours N t = N 0 x 2kt N t = 2 x 2 (3) (5) N t = 2 x 2 15 N t = 65 536 Page 16 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources 6.4 Core Practical 13: Rate of Growth of Microorganisms Your notes Rate of Growth of Microorganisms There are several ways to investigate the growth of microorganisms In this example the change in turbidity of a yeast solution will be used as a measure of the growth of microorganisms in a liquid culture over time Turbidity = cloudiness of a solution Apparatus Cloths and antimicrobial solution Conical flask Glucose solution Dried baker's yeast Bunsen burner Cotton wool Magnetic stirrer Aluminium foil Colorimeter Dropper pipette Cuvettes Optional Microscope Microscope slides with cover slips and graph paper photocopied on to acetate OR a haemocytometer Method 1. Use antimicrobial solution to sterilise the work area 2. Place 250 cm3 glucose solution into a conical flask This solution will be the liquid culture medium 3. Inoculate the glucose solution with 1.25 g dried yeast using aseptic techniques Work next to a Bunsen flame to ensure that microorganisms in the air are drawn away from the nutrient broth 4. Seal the flask using a cotton wool stopper immediately after inoculation is complete to prevent contamination 5. Swirl to mix the yeast with the glucose solution and place on a magnetic stirrer 6. While stirring continuously, loosely cover the stopper with aluminium foil and incubate the flask at 20 °C 7. Fill a cuvette with plain glucose solution and use this as a blank to calibrate the colorimeter This ensures that the colorimeter is reset to zero before each measurement is taken 8. Transfer about 3 cm3 of the yeast suspension into a cuvette using a dropping pipette 9. Use the colorimeter to measure the absorbance and record this in a suitable table against time 10. Repeat steps 7-9 at intervals over a 12 hour period Page 17 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources E.g. this could involve taking samples every 30 minutes for the first two hours and then every 2-3 hours after this 11. Plot a graph of absorbance against time Your notes The temperature or concentration of the glucose solution can be changed and the experiment repeated to investigate the effect these changes would have on the growth of the yeast Optional extension to practical Turbidity measurements are useful as the turbidity of the medium is mainly a measure of the number of living cells in suspension; however, the measurement can be affected by the presence of dead cells and other suspended particles, so other methods of cell counting can be used to back up the turbidity findings, e.g. Use a haemocytometer to estimate the number of yeast cells in the glucose solution Calculate the area of the microscope's field of view using graph paper copied on to acetate, and use the number of cells in the field of view to estimate the number of cells in the glucose solution If using the area of the microscope's field of view the following steps can be used to estimate the number of cells in the growth medium Count the number of squares of acetate graph paper visible when using the x4 objective lens and use this to calculate the area of the field of view under a x40 objective lens; this will be 0.01 of the area of the x4 objective lens Stain the yeast suspension with methylene blue and place one drop of the suspension on a microscope slide View under the x40 objective lens and count the number of yeast cells in the field of view Calculate the volume of this drop by measuring the volume of 10 drops and dividing by 10 To calculate the volume under the field of view when using the x40 objective lens, use the following formula volume = (area of field of view of x40 objective lens ÷ area of cover slip) x volume of one drop To calculate the number of yeast cells in 1 mm3 of medium the following formula can be used number of cells per mm3 = average cell count in field of view ÷ volume of field of view Note that if many cells overlap when viewed under x40 objective lens a serial dilution of the yeast suspension will be required Safety Eye protection should be worn Care should be taken around Bunsen burners Aseptic techniques should be used when transferring microorganisms Cultures should be incubated at a safe temperature for school laboratory work; 20 °C and not 37 °C Work spaces and hands should be thoroughly disinfected at the end of the experiment Cultures should be safely destroyed at the end of the experiment Page 18 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources 6.5 Comparison of Bacterial & Viral Structure Your notes Comparison of Bacterial & Viral Structure Bacteria Bacteria are single-celled prokaryotes Prokaryotic cells are much smaller than eukaryotic cells They also differ from eukaryotic cells in having A cytoplasm that lacks membrane-bound organelles Ribosomes that are smaller (70 S) than those found in eukaryotic cells (80 S) No nucleus, instead having a single circular bacterial chromosome that is free in the cytoplasm and is not associated with proteins A cell wall that contains the glycoprotein murein Murein is sometimes known as peptidoglycan In addition, many prokaryotic cells also have the following structures Loops of DNA known as plasmids Capsules This is sometimes called the slime capsule It helps to protect bacteria from drying out and from attack by cells of the immune system of the host organism Flagella (singular flagellum) Long, tail-like structures that rotate, enabling the prokaryote to move Some prokaryotes have more than one Pili (singular pilus) Thread-like structures on the surface of some bacteria that enable the bacteria to attach to other cells or surfaces Involved in gene transfer during sexual reproduction A cell membrane that contains folds known as mesosomes; these infolded regions can be the site of respiration Some bacteria are disease-causing, or pathogenic, but not all bacteria cause harm to other organisms Page 19 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources Your notes Prokaryotic cells have a peptidoglycan cell wall, no membrane-bound organelles, a circular chromosome, and 70S ribosomes Viruses Viruses are non-cellular infectious particles They are relatively simple in structure, and much smaller than prokaryotic cells Structurally they have A nucleic acid core Their genomes are either DNA or RNA, and can be single or double-stranded A protein coat called a ‘capsid’ made of repeating units known as capsomeres Page 20 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources They do not possess a plasma membrane, cytoplasm, or ribosomes Some viruses have an outer layer called an envelope formed from the membrane-phospholipids of the cell they were made in Your notes The fact that lipid envelopes are formed from the membrane of a viral host cell means that very few plant viruses have lipid envelopes Some contain proteins inside the capsid which perform a variety of functions E.g. HIV contains the enzyme reverse transcriptase which converts its RNA into DNA once it has infected a cell Viruses also contain attachment proteins, also known as virus attachment particles, that stick out from the capsid or envelope These enable the virus to attach itself to a host cell Viruses can only reproduce by infecting living cells and using the protein-building machinery of their host cells to produce new viral particles Viruses are classified on the basis of the genetic material they contain and how they replicate They can be classified into the following categories DNA viruses RNA viruses Retroviruses HIV contains RNA as its genetic material. It is surrounded by a protein capsid, as well as having an outer lipid envelope and attachment proteins DNA viruses They contain DNA as genetic material Viral DNA acts as a direct template for producing new viral DNA and mRNA for the synthesis of viral proteins Page 21 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources Examples: smallpox, adenoviruses, and bacteriophages Bacteriophages are viruses that infect bacteria, such as the λ (lambda) phage Your notes Bacteriophage viruses, such as the λ phage, are examples of DNA viruses RNA viruses They contain RNA as genetic material Most have a single strand of RNA They do not produce DNA at all Mutations are more likely to occur in RNA viruses than DNA viruses Examples: tobacco mosaic virus (TMV), ebola virus Retroviruses Special type of RNA virus that does produce DNA They contain a single strand of RNA surrounded by a protein capsid and lipid envelope Viral RNA controls the production of an enzyme called reverse transcriptase This enzyme catalyses production of viral DNA from the single strand of RNA The new viral DNA is incorporated into the host DNA using integrase enzymes where it acts as a template to produce viral proteins and RNA Page 22 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources Example: HIV (Human Immunodeficiency Virus) Your notes Page 23 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources Lytic & Latency Viruses can only reproduce within a host cell as they lack the cellular machinery to do so on their own Your notes They can enter a host cell in a variety of different ways Bacteriophages inject their genetic material into bacteria Some animal viruses enter the cell via endocytosis by fusing their viral envelope with the host cell surface membrane Plant viruses will often use a vector such as an insect to breach the cell wall Once inside the host cell one of the following pathways can occur Lysogenic Lytic Lysogenic pathway Some viruses will not immediately cause disease once they infect a host cell Viral DNA known as a provirus is inserted into the host DNA, but a viral gene coding for a repressor protein prevents the viral DNA from being transcribed and translated Every time the host DNA copies itself, the inserted viral DNA will also be copied This is called latency and the time during which it occurs is known as a period of lysogeny Viruses in a lysogenic state may become activated and enter the lytic pathway Activation may occur as a result of, e.g. host cell damage or low nutrient levels inside a cell Lytic pathway The viral genetic material is transcribed and translated to produce new viral components These components are assembled into mature viruses that accumulates inside the host cell Eventually the host cell bursts which releases large numbers of viruses, each of which can infect a new host cell Cell bursting is known as cell lysis This typically results in disease Page 24 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers  Head to www.savemyexams.com for more awesome resources Your notes The life cycle of the λ bacteriophage includes a lysogenic and a lytic pathway Page 25 of 25 © 2015-2025 Save My Exams, Ltd. · Revision Notes, Topic Questions, Past Papers